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The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons.

ABSTRACT: The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

SUBMITTER: Schmidt M

PROVIDER: S-EPMC2746555 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons.

Schmidt Mirko M Lin Shengyin S Pape Manuela M Ernsberger Uwe U Stanke Matthias M Kobayashi Kazuto K Howard Marthe J MJ Rohrer Hermann H

Developmental biology 20090227 2

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylas ...[more]

PMID: 19254708

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Project description:The sympathetic nervous system vitally regulates autonomic functions, including cardiac activity. Postganglionic neurons of the sympathetic chain ganglia relay signals from the central nervous system to autonomic peripheral targets. Disrupting this flow of information often dysregulates organ function and leads to poor health outcomes. Despite the importance of these sympathetic neurons, fundamental aspects of the neurocircuitry within peripheral ganglia remain poorly understood. Conventionally, simple monosynaptic cholinergic pathways from preganglionic neurons are thought to activate postganglionic sympathetic neurons. However, early studies suggested more complex neurocircuits may be present within sympathetic ganglia. The present study recorded synaptic responses in sympathetic stellate ganglia neurons following electrical activation of the pre- and postganglionic nerve trunks and used genetic strategies to assess the presence of collateral projections between postganglionic neurons of the stellate ganglia. Orthograde activation of the preganglionic nerve trunk, T-2, uncovered high jitter synaptic latencies consistent with polysynaptic connections. Pharmacological inhibition of nicotinic acetylcholine receptors with hexamethonium blocked all synaptic events. To confirm that high jitter, polysynaptic events were due to the presence of cholinergic collaterals from postganglionic neurons within the stellate ganglion, we knocked out choline acetyltransferase in adult noradrenergic neurons. This genetic knockout eliminated orthograde high jitter synaptic events and EPSCs evoked by retrograde activation. These findings suggest that cholinergic collateral projections arise from noradrenergic neurons within sympathetic ganglia. Identifying the contributions of collateral excitation to normal physiology and pathophysiology is an important area of future study and may offer novel therapeutic targets for the treatment of autonomic imbalance. KEY POINTS: Electrical stimulation of a preganglionic nerve trunk evoked fast synaptic transmission in stellate ganglion neurons with low and high jitter latencies. Retrograde stimulation of a postganglionic nerve trunk evoked direct, all-or-none action currents and delayed nicotinic EPSCs indistinguishable from orthogradely-evoked EPSCs in stellate neurons. Nicotinic acetylcholine receptor blockade prevented all spontaneous and evoked synaptic activity. Knockout of acetylcholine production in noradrenergic neurons eliminated all retrogradely-evoked EPSCs but did not change retrograde action currents, indicating that noradrenergic neurons have cholinergic collaterals connecting neurons within the stellate ganglion.

Project description:BackgroundThe discovery of adrenoceptors, which mediate the effects of the sympathetic nervous system neurotransmitter norepinephrine on specific tissues, sparked the development of sympathomimetics that have profound influence on skeletal muscle mass. However, chronic administration has serious side effects that preclude their use for muscle-wasting conditions such as sarcopenia, the age-dependent decline in muscle mass, force, and power. Devising interventions that can adjust neurotransmitter release to changing physiological demands will require understanding how the sympathetic nervous system affects muscle motor innervation and muscle mass, which will prevent sarcopenia-associated impaired mobility, falls, institutionalization, co-morbidity, and premature death. Here, we tested the hypothesis that prolonged heart and neural crest derivative 2 (Hand2) expression in peripheral sympathetic neurons (SNs) ameliorates sympathetic muscle denervation, motor denervation, and sarcopenia in geriatric mice.MethodsWe delivered either a viral vector encoding the transcription factor Hand2 or an empty vector (EV) driven to SNs by the PRSx8 promoter by injecting the saphenous vein in 16-month-old C57BL/6 mice that were sacrificed 10-11 months later. Studies relied on sympathetic and muscle immunohistochemistry analysed by confocal microscopy, nerve and muscle protein expression assessed by immunoblots, nerve-evoked and muscle-evoked maximal muscle contraction force, extensor digitorum longus (EDL) muscle RNA sequencing, SN real-time PCR, and tests of physical performance using an inverted-cling grip test and in an open-arena setting.ResultsExamining the mice 10-11 months later, we found that inducing Hand2 expression in peripheral SNs preserved (i) the number of neurons (EV: 0.32 ± 0.03/μm2 , n = 6; Hand2: 0.92 ± 0.08/μm2 , n = 7; P < 0.0001) and size (EV: 279 ± 18 μm2 , n = 6; Hand2: 396 ± 18 μm2 , n = 7; P < 0.0001); (ii) lumbricalis muscle sympathetic innervation (EV: 1.4 ± 1.5 μm/μm2 , n = 5; Hand2: 12 ± 1.8 μm/μm2 , n = 5; P < 0.001); (iii) tibialis anterior, gastrocnemius, EDL, and soleus muscles weight and whole-body strength (EV: 48 ± 6.4 s, n = 6; Hand2: 102 ± 6.8 s, n = 6; P < 0.001); (iv) EDL type IIb, IIx, and II/IIx and soleus type I, IIa, IIx, IIa/IIx, and IIb/IIx myofibre cross-sectional area; (v) nerve-evoked (EV: 16 ± 2.7 mN; Hand2: 30 ± 4.4 mN; P < 0.001) and muscle-evoked (EV: 24 ± 3.8 mN, n = 5; Hand2: 38 ± 3.0 mN, n = 8; P < 0.001) muscle force by 150 Hz-3 s pulses; and (vi) motor innervation assessed by measuring presynaptic/postsynaptic neuromuscular junction area overlay.ConclusionsPreserving Hand2 expression in SNs from middle-aged to very old mice attenuates decreases in muscle mass and force by (i) maintaining skeletal muscle sympathetic and motor innervation, (ii) improving membrane and total acetylcholine receptor stability and nerve-evoked and muscle-evoked muscle contraction, (iii) preventing the elevation of inflammation and myofibrillar protein degradation markers, and (iv) increasing muscle autophagy.

Project description:Noradrenaline is a neurotransmitter involved in general arousal, selective attention, memory, inflammation, and neurodegeneration. The purpose of this work was to delineate noradrenergic neurons in vivo by T1-weighted MRI with magnetization transfer (MT). In the brainstem of human and mice, MRI identified the locus coeruleus, dorsal motor vagus nucleus, and nucleus tractus solitarius. Given (1) the long T1 and low magnetization transfer ratio for the noradrenergic cell groups compared to other gray matter, (2) significant correlation between MT MRI signal intensity and proton density, and (3) no correlation between magnetization transfer ratio (or R1) and iron, copper, or manganese in human brain, the high MRI signal of the noradrenergic neurons must be attributed to abundant water protons interacting with any T1-shortening paramagnetic ions in active cells rather than to specific T1-shortening molecules. The absence of a high MRI signal from the locus coeruleus of Ear2(-/-) mice lacking noradrenergic neurons confirms that cell bodies of noradrenergic neurons are the source of the bright MRI appearance. The observation of this high signal in DBH(-/-) mice, in 3-week-old mice, and in mice under hyperoxia/hypercapnia/hypoxia together with the general absence of neuromelanin (NM) in noradrenergic neurons of young rodents further excludes that it is due to NM, dopamine β-hydroxylase, their binding to paramagnetic ions, blood inflow, or hemoglobin. Instead, these findings indicate a high density of water protons whose T1 is shortened by paramagnetic ions as the relevant source of the high MRI signal. In the brain of APP/PS1/Ear2(-/-) mice, a transgenic model of Alzheimer's disease, MRI detected noradrenergic neuron loss in the locus coeruleus. Proton magnetic resonance spectroscopy revealed that a 60-75% reduction of noradrenaline is responsible for a reduction of N-acetylaspartate and glutamate in the hippocampus as well as for a shortening of the water proton T2 in the frontal cortex. These results suggest that a concurrent shortage of noradrenaline in Alzheimer's disease accelerates pathologic processes such as inflammation and neuron loss.

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The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons.

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The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons.

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